Application of pear transcription factor PbrWRKY83 and PbrDHAR5 genes in enhancing low-temperature stress resistance of fruits

Abstract

The invention discloses an application of pear transcription factor PbrWRKY83 and PbrDHAR5 genes in enhancing the low-temperature stress resistance of fruits. The nucleotide sequence of the pear WRKY family transcription factor PbrWRKY83 is as shown in SEQ ID NO.3 in a sequence table, and the amino acid sequence of the gene coded protein is as shown in SEQ ID NO.4. The invention further discloses a preparation method of the pear WRKY family transcription factor PbrWRKY83. The invention discloses the transcription factor PbrWRKY83 for regulating and controlling the PbrDHAR5 gene expression of the pear fruit for the first time. The invention illustrates a W-box element (P1:-453)-(-488) in a PbrWRKY83 gene promoter and a PbrDHAR5 gene promoter, and also illustrates a W-box element (P1:-453)-(-488) in a PbrDHAR5 gene promoter. The invention provides an action mechanism for regulating the AsA oxidation-reduction state of the pear and further enhancing the low-temperature stress resistance of the fruit through interaction of the (P2: (-1233)-(-1228)), and a theoretical and practical basis is provided for cultivating a high-quality pear variety capable of resisting tiger skin disease.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and genetic engineering, and specifically relates to a pear transcription factor PbrWRKY8 that regulates the AsA redox state by regulating the expression of a key gene PbrDHAR5 in the ascorbic acid-glutathione cycle (AsA-GSH) to thereby enhance the ability of fruit to resist low temperature stress. Background Art

[0002] To meet market demand throughout the year, harvested pear (Pyrus spp.) fruits are usually stored in cold rooms for several months. However, this treatment can cause brown scald disease, which manifests as brown or black patches on the skin. It was reported that between 2006 and 2018, the 'Dangshan Pear' suffered at least 154 million yuan in economic losses each year due to brown scald disease. Over the past half century, efforts have been made to screen effective physical and chemical treatments to alleviate brown scald symptoms, such as 1-MCP fumigation, diphenylamine (DPA) impregnation, proline soaking, and controlled atmosphere (CA) storage.

[0003] Cellular redox homeostasis plays a key role in the development of pear fruit tiger skin disease. As a highly abundant metabolite, ascorbic acid (AsA) and its redox state (AsA / DHA) are involved in the plant response to abiotic stress. In addition to its antioxidant effect, AsA is also involved in the complex and orderly adaptation to environmental stress through redox signaling, transcriptional regulation, and protein function modification; on the other hand, the AsA redox state can serve as a sensor of the redox state in plant cells, sensing and coordinating its actions, such as reactive oxygen species (ROS) scavenging and its signal transduction. Studies have shown that ascorbic acid levels are positively correlated with tiger skin disease resistance; exogenous ascorbic acid treatment can alleviate the occurrence of fruit tiger skin disease. These findings suggest the role of AsA and its redox state in the occurrence of tiger skin disease.

[0004] The level of ascorbic acid (AsA) and its redox state in plants depend on the balance between ascorbic acid biosynthesis and recycling. In higher plants, the main pathway for ascorbic acid accumulation is the D-mannose / L-galactose pathway, in which GDP-L-galactose phosphorylase (GGP) is the rate-limiting enzyme. After ascorbic acid is formed in mitochondria, it is transported to other subcellular organelles, where it accepts electrons from various free radicals to generate its oxidized forms, dehydroascorbic acid (DHA) and monodehydroascorbic acid (MDHA); while DHA and MDHA can be reduced to AsA by the catalysis of DHA reductase (DHAR) and MDHA reductase (MDHAR) in the ascorbate-glutathione (AsA-GSH) cycle. Heterologous overexpression of the chloroplast PbrDHAR2 gene (Gene ID: Pbr016672.1) increased AsA levels and its redox state in tomato, thereby alleviating salinity and low temperature stress. Similar phenomena were observed in potato by overexpression of the AtDHAR1 gene.

[0005] For horticultural fruits, the expression of structural genes is regulated by transcription factors (TFs) at the transcriptional level. These transcription factors regulate the expression of downstream genes by interacting with the corresponding cis-acting elements in their promoters. The WRKY transcription factor PbrWRKY40 from white pear can bind to the V-type H + -ATPase (VHA-B1) promoter, activates its expression, and improves salt tolerance in Arabidopsis and pear callus. PbrMYB5 upregulates the expression of PbrDHAR2 gene, increases the level of AsA and its redox state, thereby reducing the sensitivity of white pear to low temperature. However, until recently, the mechanism of the role of AsA and its redox state in the occurrence of tiger skin disease has not been elucidated. Summary of the invention

[0006] The object of the present invention is to provide an application of a PbrDHAR5 gene or a biological material related to the PbrDHAR5 gene in regulating the redox state of AsA and thus enhancing the ability of fruit to resist low temperature stress.

[0007] Another object of the present invention is to provide an application of a WRKY family transcription factor PbrWRKY83 or a biological material related to the WRKY family transcription factor PbrWRKY83 in regulating the redox state of AsA and thereby enhancing the ability of fruit to resist low temperature stress.

[0008] Another object of the present invention is to provide a method for enhancing the resistance of fruit to low temperature stress. The WRKY family transcription factor PbrWRKY83 specifically binds to the W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228)) in the upstream promoter of the PbrDHAR5 gene, promoting the transcription of the PbrDHAR5 gene.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention claims the use of the PbrDHAR5 gene or biological materials related to the PbrDHAR5 gene in regulating the redox state of ascorbic acid (AsA) and thereby enhancing the ability of fruit to resist low temperature stress. The nucleotide sequence of the PbrDHAR5 gene is shown in SEQ ID NO.1.

[0011] Furthermore, the biological material related to the PbrDHAR5 gene is at least one of (a1) to (a7):

[0012] (a1) PbrDHAR5 protein with an amino acid sequence as shown in SEQ ID NO.2;

[0013] (a2) an expression cassette containing the PbrDHAR5 gene;

[0014] (a3) a recombinant vector containing the PbrDHAR5 gene, or a recombinant vector containing the expression cassette described in (a2);

[0015] (a4) a recombinant microorganism containing the PbrDHAR5 gene, or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3);

[0016] (a5) a transgenic plant cell line containing the PbrDHAR5 gene, or a transgenic plant cell line containing the expression cassette described in (a2), or a transgenic plant cell line containing the recombinant vector described in (a3);

[0017] (a6) transgenic plant tissue containing the PbrDHAR5 gene, or transgenic plant tissue containing the expression cassette described in (a2), or transgenic plant tissue containing the recombinant vector described in (a3);

[0018] (a7) A transgenic plant organ containing the PbrDHAR5 gene, or a transgenic plant organ containing the expression cassette described in (a2), or a transgenic plant organ containing the recombinant vector described in (a3).

[0019] Furthermore, the above application is to overexpress the PbrDHAR5 gene to regulate the redox state of ascorbic acid and thus enhance the fruit's resistance to low temperature stress.

[0020] In the second aspect, the present invention seeks to protect the use of WRKY family transcription factor PbrWRKY83 or biological materials related to WRKY family transcription factor PbrWRKY83 in regulating the redox state of ascorbic acid (AsA) and thereby enhancing the ability of fruit to resist low temperature stress. The amino acid sequence of the transcription factor PbrWRKY83 is shown in SEQ ID NO.4.

[0021] Furthermore, the biological material related to the WRKY family transcription factor PbrWRKY83 is at least one of (b1) to (b7):

[0022] (b1) gene PbrWRKY83 encoding the WRKY family transcription factor PbrWRKY83;

[0023] (b2) an expression cassette containing the gene PbrWRKY83 described in (b1);

[0024] (b3) a recombinant vector containing the gene PbrWRKY83 described in (b1), or a recombinant vector containing the expression cassette described in (b2);

[0025] (b4) a recombinant microorganism containing the gene PbrWRKY83 described in (b1), or a recombinant microorganism containing the expression cassette described in (b2), or a recombinant microorganism containing the recombinant vector described in (b3);

[0026] (b5) a transgenic plant cell line containing the gene PbrWRKY83 described in (b1), or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3);

[0027] (b6) transgenic plant tissue containing the gene PbrWRKY83 described in (b1), or transgenic plant tissue containing the expression cassette described in (b2), or transgenic plant tissue containing the recombinant vector described in (b3);

[0028] (b7) A transgenic plant organ containing the gene PbrWRKY83 described in (b1), or a transgenic plant organ containing the expression cassette described in (b2), or a transgenic plant organ containing the recombinant vector described in (b3).

[0029] Furthermore, the nucleotide sequence of the gene PbrWRKY83 encoding the WRKY family transcription factor PbrWRKY83 in (b1) is shown as SEQ ID No.3.

[0030] Furthermore, the above application is that the WRKY family transcription factor PbrWRKY83 interacts with the PbrDHAR5 gene to regulate the redox state of ascorbic acid and thus enhance the fruit's resistance to cold stress. Furthermore, the WRKY family transcription factor PbrWRKY83 promotes the transcription of the PbrDHAR5 gene by binding to the WRKY binding site (W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228))) in the PbrDHAR5 gene promoter, regulates the redox state of ascorbic acid and thus enhances the fruit's resistance to cold stress; the P1 binding site sequence is (+ / -)TTGACT / AGTCAA; the P2 binding site sequence is (+ / -)TTGACT / AGTCAA. The nucleotide sequence of the PbrDHAR5 gene promoter is shown in SEQ ID NO.5.

[0031] In a third aspect, the present invention claims a method for enhancing the ability of Rosaceae fruit to resist low temperature stress, which uses any one of the following methods (c1)-(c2) or a combination of two methods to promote ascorbic acid accumulation and thus enhance the ability of the fruit to resist low temperature stress;

[0032] (c1) overexpressing the gene PbrWRKY83 encoding the transcription factor PbrWRKY83 or / and increasing the protein level or activity of the transcription factor PbrWRKY83;

[0033] (c2) overexpressing the PbrDHAR5 gene or / and increasing the protein level or activity of the PbrDHAR5 protein encoded by the PbrDHAR5 gene;

[0034] The nucleotide sequence of the gene PbrWRKY83 encoding the transcription factor PbrWRKY83 is shown in SEQ ID No.3; the amino acid sequence of the transcription factor PbrWRKY83 is shown in SEQ ID No.4; and the nucleotide sequence of the PbrDHAR5 gene is shown in SEQ ID No.1.

[0035] The fruit described in the present invention is a fruit of a fruit tree of the Rosaceae family, such as pear, or a fruit of a vegetable of the Solanaceae family, such as tomato.

[0036] Research process of the technical solution of the present invention:

[0037] The redox state of AsA was determined to be involved in the pathogenesis of the 'Dangshan Pear' fruit chafing disease. The expression profiles of related genes in the AsA-GSH cycle pathway and their correlation with ascorbic acid content, morbidity and disease index during the pathogenesis of the 'Dangshan Pear' chafing disease were further analyzed, and PbrDHAR5 was screened out, which may be involved in the pathogenesis of the 'Dangshan Pear' chafing disease by regulating the redox state of AsA.

[0038] The recombinant plasmid pBI221-PbrDHAR5-GFP was constructed and transformed into tobacco leaves and Arabidopsis protoplasts. Using AtDHAR2-mcherry as a marker, laser confocal microscopy revealed that PbrDHAR5 was located in the cytoplasm and nucleus.

[0039] The pCAMBIA1300-PbrDHAR5-GFP overexpression vector and the pTRV2-PbrDHAR5 silencing vector were constructed and transformed into Agrobacterium GV3101, and the positive strains were collected and injected into the pear peel. In addition, tomato explants were infected with a vector carrying the PbrDHAR5 gene overexpression vector (pCAMBIA1300-PbrDHAR5-GFP), and positive homozygous strains were obtained by hygromycin resistance test and PCR detection; tomato fruits of the positive homozygous strains of the F2 generation were harvested (fruits 35 days after flowering), refrigerated at 4°C for 10 days, and then placed at 20°C for 7 days.

[0040] The relative expression level of PbrDHAR5 gene in pear peel tissue was detected by RT-qPCR technology. It was found that the expression level of this gene was significantly increased in the peel of the fruit with transient overexpression of PbrDHAR5 gene, while the expression level of this gene was significantly decreased in the peel of the fruit with transient silencing of PbrDHAR5.

[0041] Using high performance liquid chromatography (HPLC), it was found that the AsA redox state in the peel tissue of pear and tomato fruits with transient overexpression of the PbrDHAR5 gene was significantly improved, while the opposite phenomenon was observed in the peel of fruits with transient silencing of the PbrDHAR5 gene. Further research found that overexpression of the PbrDHAR5 gene significantly improved the AsA redox state of tomato fruit and its resistance to low temperature.

[0042] Transcriptome analysis revealed a total of 38 differentially expressed TFs; of these, 6 were strongly positively correlated with PbrDHAR5 during cold storage of ‘Dangshan Pear’ fruit (fold change ≥ 1.5, FDR < 0.05; correlation coefficient ≥ 0.8). Since Pbr008639.1 had the highest correlation with PbrDHAR5 expression, it was named PbrWRKY83 and further studied. With the help of the PlantRegMap database, PbrWRKY83 may interact with two W-box elements in the PbrDHAR5 promoter (sequence 1502 bp upstream of the transcription start site (ATG)).

[0043] The recombinant plasmid pSAK277-PbrWRKY83 was constructed, and the reporter vectors (pGreenII 0800-PbrDHAR5pro-LUC (full-length promoter), p ... frag1 -LUC, pGreenII 0800-PbrDHAR5pro frag2 -LUC, pGreenII 0800-PbrDHAR5pro mut -LUC) were transformed into Agrobacterium GV3101 and GV3101(psoup) respectively. Agrobacterium strains carrying pSAK277-PbrWRKY83 and the reporter vector were used to co-infect tobacco leaves, and the activities of firefly luciferase and Renilla luciferase were detected. It was found that pSAK277-PbrWRKY83&pGreenII 0800-PbrDHAR5pro-LUC (or pSAK277-PbrWRKY83&pGreenII 0800-PbrDHAR5pro frag1 -LUC) co-transformed tobacco leaves had a significantly higher Luc / Ren ratio than the control group, while pSAK277-PbrWRKY83&pGreenII 0800-PbrDHAR5pro frag2 -LUC(or pSAK277-PbrWRKY83&pGreenII 0800-PbrDHAR5pro mut There was no significant difference in the Luc / Ren ratio of tobacco leaves co-transformed with WT-LUC and its control group.

[0044] The prey vector PbrWRKY83-AD was constructed, and the bait vector (PbrDHAR5pro) was constructed with the PbrDHAR5 promoter fragment containing the wild-type W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228)) or its mutant ((TTGACC / T→TTTAGC / T) S1 -pAbAi and PbrDHAR5pro S2 -pAbAi); Matchmaker Gold Yeast One-Hybrid Library Screening System was used to detect that PbrWRKY83 could bind to a promoter fragment containing a W-box element (PbrDHAR5pro S1 -pAbAi and PbrDHAR5pro S2 -pAbAi) binding.

[0045] The recombinant plasmid pCAMBIA1300-PbrWRKY83-GFP was constructed and transformed into pear callus. Pear callus carrying pCAMBIA1300-GFP was used as a control. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis was performed on the PbrDHAR5 promoter fragment containing the W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228)), and it was found that PbrWRKY83 could bind to the promoter fragment containing the W-box element.

[0046] Combined with further electrophoretic mobility shift assay (EMSA) results, it was concluded that PbrWRKY83 activated the expression of PbrDHAR5 gene by binding to the W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228)) in the upstream promoter of the gene.

[0047] The recombinant plasmid pBI221-PbrWRKY83-GFP was constructed and transformed into tobacco leaves and Arabidopsis protoplasts. AtH2B-mcherry was used as a nuclear marker. Laser confocal microscopy revealed that PbrWRKY83 was located in the cell nucleus.

[0048] The pCAMBIA1300-PbrWRKY83-GFP overexpression vector and the pTRV2-PbrWRKY83 silencing vector were constructed and transformed into Agrobacterium GV3101, and the positive strains were collected and injected into the pear peel. In addition, tomato explants were infected with the PbrWRKY83 gene overexpression vector (pCAMBIA1300-PbrWRKY83-GFP), and positive homozygous strains were obtained by hygromycin resistance test and PCR detection; the tomato fruits of the F2 generation positive homozygous strains (fruits 35 days after flowering) were harvested, refrigerated at 4°C for 10 days, and then placed at 20°C for 7 days.

[0049] The relative expression levels of PbrWRKY83 and PbrDHAR5 genes in pear peel tissue were detected by RT-qPCR technology. It was found that the expression levels of PbrWRKY83 and PbrDHAR5 genes in the peel of pear with transient overexpression of PbrWRKY83 gene were significantly increased, while the expression levels of PbrWRKY83 and PbrDHAR5 genes in the peel of pear with transient silencing of PbrWRKY83 gene were significantly decreased.

[0050] Using high performance liquid chromatography (HPLC), it was found that the AsA redox state in the peel tissue of pear and tomato fruits with transient overexpression of the PbrWRKY83 gene was significantly improved, while the opposite phenomenon was observed in the peel of fruits with transient silencing of the PbrWRKY83 gene. Further studies found that overexpression of the PbrWRKY83 gene significantly improved the AsA redox state of tomato fruit and its resistance to low temperature.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. The transcription factor PbrWRKY83 that regulates the expression of the pear PbrDHAR5 gene was revealed for the first time.

[0053] 2. The present invention clarifies the mechanism of interaction between PbrWRKY83 and the W-box element in the promoter of PbrDHAR5 gene (P1: (-453)-(-488); P2: (-1233)-(-1228)) in regulating the redox state of AsA and thus enhancing the fruit's resistance to low temperature stress, providing a theoretical and practical basis for breeding high-quality pear varieties resistant to tiger skin disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0055] Figure 1 The redox state of AsA is involved in the pathogenesis of the tiger skin disease in 'Dangshan Pear' fruit.

[0056] Among them, (a) appearance quality; (b) changes in the content of AsA-related metabolites; (c) correlation analysis. The fruits of 'Dangshan Pear' were randomly divided into three treatments: H2O (control), DPA immersion and 1-MCP fumigation, and stored at 0.5℃ for 0, 60, 120, and 180 days, respectively. Samples were taken after 7 days of storage at room temperature. The color scale represents the normalized log2 transformation (the average of three biological replicates + 1), where red represents high levels, blue represents low levels, and white represents medium levels. The Pearson correlations between different attributes are visualized as heat maps, where red lines represent positive correlations, while blue lines represent negative correlations.

[0057] Figure 2 Screening of the key candidate gene PbrDHAR5 that regulates the redox state of AsA.

[0058] Among them, (a) AsA metabolic pathway; (b) Changes in the activities and gene expression of related enzymes in the AsA-GSH cycle during the cold storage of ‘Dangshan Pear’ fruit. (bi) Cytoplasmic dehydroascorbate reductase (cytDHAR) and cytoplasmic monodehydroascorbate reductase (cytMDHAR) activities. Data are the average of three biological replicates; samples with the same lowercase letter above the vertical bar did not differ significantly at the same sampling time (p<0.05). (b-ii) Gene expression profiles and their correlation with quality and physiological and biochemical characteristics. Data are from transcriptome assays and represent the average of three biological replicates. The color scale represents the standardized log2 transformation (mean FPKM+1), where red indicates high levels, blue indicates low levels, and white indicates medium levels. The Pearson correlations between different attributes are visualized as heat maps, where extremely strong (or strong) positive correlations are connected with red (or light red) lines, and extremely strong (or strong) negative correlations are connected with blue (or light blue) lines. The fruits of 'Dangshan Pear' were randomly divided into three treatments: H2O (control), DPA immersion, and 1-MCP fumigation. The fruits were stored at 0.5℃. The fruits were sampled every 60 days and then stored at ambient temperature for 7 days.

[0059] Figure 3 Functional verification of PbrDHAR5 gene.

[0060] Among them, (a) subcellular localization of PbrDHAR5. AtDHAR2-mcherry was used as a marker. (b) Effect of transient transformation of 'Dangshan Pear' on the redox state of AsA in fruit. (bi) Transient overexpression of PbrDHAR5 gene. The fruit of 'Dangshan Pear' transformed with empty pCAMBIA1300 vector was used as control. (b-ii) Transient silencing of PbrDHAR5 gene. The fruit of 'Dangshan Pear' co-transformed with empty pTRV2 and pTRV1 was used as control. The expression level of PbrDHAR5 gene in control pear was set to 1.0. (c) Effect of overexpression of PbrDHAR5 gene on the redox state of AsA in tomato. (ci) Appearance quality changes and chilling injury index. (c-ii) PbrDHAR5 gene expression level and cytDHAR activity. (c-iii) AsA redox state and H2O2 content. Tomato fruits under 35 DAFB, including those from wild-type (control) and overexpression (OE) plants, were collected and stored at 20°C for 7 days after low-temperature treatment at 4°C for 10 days. The expression level of PbrDHAR5 in OE-5 fruits was set to 1.0. Data are the average of three biological replicates; samples with the same lowercase letter above the vertical bar had no significant difference within the same sampling time (p<0.05).

[0061] Figure 4 Screening upstream regulatory factors that regulate PbrDHAR5 gene expression.

[0062] Among them, (a) the number of differentially expressed TFs. (b) the expression profiles of differentially expressed TFs and their correlation with the abundance of PbrDHAR5 mRNA. The fruits of 'Dangshan Pear' were randomly divided into three treatments: H2O (control), DPA immersion and 1-MCP fumigation. They were stored at 0.5℃ for 0, 60, 120 and 180 days, respectively. The samples were taken after 7 days of storage at room temperature. Using DESeq2_EBSeq software, foldchange≥1.5 and FDR<0.05 were used as the criteria to screen out TFs whose mRNA abundance in the control group was always higher or lower than that in the 1-MCP / DPA treated pears during cold storage. The data are from transcriptome assays and represent the average of three biological replicates. The color scale represents the standardized log2 transformation (mean FPKM+1), where red indicates high level, blue indicates low level, and white indicates medium level. The Pearson correlations between different attributes are visualized as heat maps, where extremely strong (or strong) positive correlations are associated with red (or light red) lines, and extremely strong (or strong) negative correlations are associated with blue (or light blue) lines. (c) Detailed information on two W-box elements in the promoter of the PbrDHAR5 gene. The W-box elements in the promoter of the PbrDHAR5 gene were analyzed using the PlantCARE database. Figure 5PbrWRKY83 regulates the expression of PbrDHAR5 gene.

[0063] Among them, (a) Dual luciferase reporter assay. Transformants containing empty pSAK277 and each reporter were used as controls. (b) Y1H assay. Yeast cells co-transformed with pGAD-p53 and p53-AbAi were used as positive controls, and yeast cells co-transformed with empty AD vector and each bait were used as negative controls. (c) ChIP-PCR analysis. Callus overexpressing the empty vector was used as a negative control. (d) EMSA assay. An unlabeled PbrDHAR5 promoter fragment containing a W-box element was used as a competitive probe. The presence and absence of His protein, His-PbrWRKY83 protein, labeled probe, or competitive probe were indicated by "+" and "-", respectively. The concentrations of the competitive probe were 50 times (50x) and 100 times (100x) of the labeled probe, respectively. The data are the average of three biological replicates; the vertical bars are marked with the same lowercase letters, and the differences between samples were not significant (p<0.05).

[0064] Figure 6 Functional verification of PbrWRKY83.

[0065] Among them, (a) Subcellular localization of PbrWRKY83. AtH2B-mcherry was used as a nuclear marker. (b) Effect of transient transformation of 'Dangshan Pear' on the redox state of AsA in the fruit. (bi) Transient overexpression of PbrWRKY83 gene. The fruit of 'Dangshan Pear' transformed with the empty pCAMBIA1300 vector was used as the control. (b-ii) Transient silencing of PbrWRKY83 gene. The fruit of 'Dangshan Pear' co-transformed with empty pTRV2 and pTRV1 was used as the control. The expression level of PbrWRKY83 gene in the control pear was set to 1.0. (c) Effect of overexpression of PbrWRKY83 in tomato on the redox state of AsA. (ci) Changes in appearance quality and chilling injury index. (c-ii) PbrWRKY83 gene expression level and cytDHAR activity. (c-iii) AsA redox state and H2O2 content. Tomato fruits under 35 DAFB, including those from wild-type (control) and overexpression (OE) plants, were collected and stored at 20°C for 7 days after low-temperature treatment at 4°C for 10 days. The expression level of PbrWRKY83 in OE-2 fruits was set to 1.0. Data are the means of three biological replicates; within the same sampling time, there is no statistically significant difference between samples marked with the same lowercase letter (p<0.05).

[0066] Figure 7 Schematic diagram of the process by which the PbrWRKY83-PbrDHAR5 module regulates the occurrence of tiger skin disease in pear fruit. DETAILED DESCRIPTION

[0067] The present invention is described in detail below in conjunction with specific embodiments. The examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following embodiments are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

[0068] Example 1 Mining the key gene PbrDHAR5 involved in the pathogenesis of pear tiger skin disease in the ascorbic acid metabolic pathway

[0069] 1. Collection of Tissue Samples

[0070] This study used 'Dangshan Pear' fruits of uniform size, free of mechanical damage and pests and diseases as experimental materials. The harvested 'Dangshan Pear' (Pucheng, Shaanxi, 2016) was refrigerated at 0.5℃ for 180 days. Every 60 days, some fruits were placed at 20℃ to simulate shelf life for 7 days.

[0071] 2. Determination of the incidence and disease index of tiger skin disease

[0072] The fruits are divided into 4 grades according to the percentage of the fruit's tiger skin area to the total area of ​​the fruit: Grade 0: no tiger skin on the fruit; Grade 1: tiger skin area ≤ 25%; Grade 2: 25% < tiger skin area ≤ 50%; Grade 3: tiger skin area > 50%.

[0073] The incidence of tiger skin disease = number of diseased fruits / number of surveyed fruits × 100%.

[0074] The disease index of tiger skin disease = Σ(number of diseased fruits × diseased fruit level) / (number of surveyed fruits × 3).

[0075] 3. Determination of ascorbic acid content in tissues

[0076] High performance liquid chromatography (HPLC) was used to determine the ascorbic acid content in the peel tissue. 0.5 g of tissue was homogenized in 2.0 mL of oxalic acid (w / v, g / 100 ml, 0.1%) and then centrifuged at 50,000 g for 15 min at 4 °C. Subsequently, the supernatant was filtered through a 0.45 μm filter and the AsA content was determined using an Ultimate 3000 high performance liquid chromatography (HPLC) system. 20 mg·L -1 T-AsA was measured after addition of dithiothreitol (DTT), and the difference between T-AsA and AsA was taken as the DHA content.

[0077] 4. Enzyme Activity Assay

[0078] 0.2 g of sample was homogenized in the corresponding extraction buffer and then centrifuged at 50000g for 15 min. After collecting the supernatant, the activity of cytosolic dehydroascorbate reductase (cytDHAR) (DHAR-1-w, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) or cytosolic monodehydroascorbate reductase (cytMDHAR) (MDHAR-1-w, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) was determined according to the formula of the relevant detection kit. The protein concentration in the supernatant was determined using a bicinchoninic acid protein detection kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0079] 5. Transcriptome Analysis

[0080] Total RNA was extracted from pear peel tissue using the EASYspin Plus Plant RNA Rapid Extraction Kit (RN3802, Aidelai, Beijing). The purity, concentration, and integrity of the sample RNA were identified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The transcriptome library was then constructed using the NEB Next UltraTM RNA Library Prep Kit for Illumina (E7770, NEB, USA). The constructed library was then sequenced using the HiSeq X-ten sequencing platform. After removing low-quality reads, clean reads were obtained for subsequent data analysis. HISAT2 software was used to compare with the Dangshan Pear genome database (http: / / peargenome.njau.edu.cn / ), and gene expression levels (FPKM) were calculated. Transcriptome sequencing and analysis were performed by Biomarker Biotechnology Co., Ltd. (Beijing, China).

[0081] 6. Screening of key candidate genes involved in the pathogenesis of pear tiger skin disease in the AsA-GSH cycle

[0082] Transcriptome annotation was used to discover genes involved in the AsA-GSH cycle. On this basis, the expression profiles of genes involved in the AsA-GSH cycle during the disease process of Dangshan Pear fruit and their correlation with AsA redox state, H2O2, enzyme activity, disease index and incidence were analyzed to screen out genes involved in the pathogenesis of pear tiger skin disease in the ascorbic acid metabolic pathway.

[0083] Experimental results: Figure 1As shown in the results, tiger skin disease appeared in the epidermal tissue of 'Dangshan Pear' fruit after low temperature treatment for 120 days, and spread to all fruits at 180 days. Exogenous 1-MCP and DPA treatment inhibited the formation of H2O and alleviated the occurrence of tiger skin. During this process, the contents of T-AsA and DHA increased with the decrease of AsA redox state. 1-MCP and DPA treatment inhibited these changes. Further analysis found that the AsA redox state was significantly negatively correlated with the progression of the disease (correlation coefficient ≤-0.6).

[0084] In plants, the redox state of AsA is controlled by the AsA-GSH cycle, and DHAR and MDHAR are key enzymes for AsA regeneration. As the low-temperature treatment time of Dangshan Pear fruit increased, the activities of cytMDHAR and cytDHAR increased steadily; exogenous 1-MCP and DPA treatment inhibited the activity of cytDHAR, but enhanced the activity of cytMDHAR ( Figure 2 ). According to the transcriptome results, 24 of the 25 genes in the pear AsA-GSH cycle were transcribed during cold storage, and the expression patterns were different. Further analysis showed that the expression levels of PbrDHAR5 and PbrAPX2 / 14 genes gradually increased, and were strongly positively correlated with H2O2 content, cytDHAR activity, and morbidity / disease index, and negatively correlated with AsA redox state (absolute correlation coefficient ≥ 0.6); while PbrAPX10 and PbrMDHAR7 / 8 genes showed the opposite results (absolute correlation coefficient ≥ 0.6). Combined with previous studies, PbrDHAR5 may participate in the pathogenesis of 'Dangshan Pear' fruit by regulating the AsA redox state.

[0085] Example 2 Functional verification of PbrDHAR5 gene

[0086] 1. Subcellular localization of the protein encoded by PbrDHAR5 gene

[0087] The coding sequence of the PbrDHAR5 gene (excluding the stop codon) was amplified using the fruit cDNA of 'Dangshan Pear' as a template (primer sequences are shown in Table 1 in the attached table), inserted into the pBI221-GFP vector, and transformed into tobacco leaves. AtDHAR2-mcherry was used as a marker, and the fluorescence signal was observed using a confocal microscope (Leica Microsystems, Germany). The CDS sequence of the PbrDHAR5 gene is shown in SEQ ID No. 1; the protein sequence encoded by the PbrDHAR5 gene is shown in SEQ ID No. 2.

[0088] 2. Transient overexpression of PbrDHAR5 gene in pear peel

[0089] Using the fruit cDNA of 'Dangshan Pear' as a template, the coding sequence of the PbrDHAR5 gene was amplified (primer sequences are shown in Table 1 in the attached table), inserted into the pCAMBIA1300 plasmid, and transformed into Agrobacterium GV3101. After PCR identification, positive strains were selected for propagation and cultured until OD 600 When the OD value was 0.6-0.8, the cells were collected; the cells were resuspended in infection solution (10 mM MgCl2, 10 mM MES and 200 mM acetosyringone), and after induction for 4 h under dark conditions, the OD value was adjusted. 600 The bacterial solution was injected into the peel tissue of Dangshan Pear slowly and evenly with a syringe, and the skin tissue was kept away from light for 24 hours after injection. The peel tissue of the injection site was collected 3 days after injection for subsequent determination. At the same time, fruits of similar size without pests and mechanical injuries were selected and injected with Agrobacterium carrying pCAMBIA1300 as a control.

[0090] 3. Transient silencing of the PbrDHAR5 gene in pear peel

[0091] Using the fruit cDNA of 'Dangshan Pear' as a template, the coding sequence of the PbrDHAR5 gene of about 250 bp was amplified (primer sequences are shown in Table 1 in the attached table), inserted into the pTRV2 vector, and transformed into Agrobacterium GV3101. After PCR identification, positive strains were selected for propagation and cultured until OD 600 When the OD value is 0.6-0.8, the cells are collected; the cells are resuspended in infection solution (10 mM MgCl2, 10 mM MES and 200 mM acetosyringone), and after 4 h of dark induction, the OD value is adjusted. 600 to 1.0. The bacterial solutions carrying the recombinant plasmids pTRV2-PbrDHAR5 and pTRV1 were mixed at a volume ratio of 1:1 and injected into the peel tissue of 'Dangshan Pear'. After injection, the mixture was kept away from light for 24 hours. The flesh of the injected part was collected 3 days after injection for subsequent determination. The peel tissue co-injected with bacterial solutions carrying pTRV2 empty vector and pTRV1 was used as a control.

[0092] 4. Obtaining transgenic tomato fruits and testing low temperature resistance

[0093] The constructed pCAMBIA1300-PbrDHAR5 overexpression vector was transferred into Agrobacterium GV3101 by Agrobacterium-mediated genetic transformation, and tomato cotyledons were infected. Tissue culture seedlings were obtained by callus induction, resistance induction differentiation and rooting culture. The T2 generation overexpression seeds were tested for hygromycin resistance, and the positive transgenic homozygous strains were verified by PCR technology. The seeds of the T2 homozygous strain were planted, and the fruits were harvested 35 days after flowering, refrigerated at 4°C for 10 days and then placed at 20°C for 7 days. The wild-type tomato fruits 35 days after flowering were used as the control, and the chilling injury index was calculated according to the fruit maturity stage = Σ (number of fruits × fruit maturity stage) / total number of surveyed fruits (0 = red maturity stage, 1 = pink stage, 2 = color change stage, 3 = color breaking stage, 4 = green maturity stage).

[0094] 5. Analysis of PbrDHAR5 gene expression in tissues (qRT-PCR)

[0095] Total RNA was extracted from the tissue using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.), and the extracted total RNA was reverse transcribed to synthesize cDNA. The expression level of the PbrDHAR5 gene was detected using Light Cycler 480SYBR GreenIMaster (4887352001, Roche) reagent (primer sequences are shown in Table 1 in the attached table). PbrTub was used as an internal reference gene.

[0096] 6. Enzyme Activity Assay

[0097] 0.2 g of sample was homogenized in the corresponding extraction buffer and then centrifuged at 50000 g for 15 min. After collecting the supernatant, cytosolic dehydroascorbate reductase (cytDHAR) (DHAR-1-w, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) was determined according to the formula of the relevant detection kit. The protein concentration in the supernatant was determined using a bicinchoninic acid protein detection kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0098] 7. Determination of ascorbic acid content in tissue

[0099] High performance liquid chromatography (HPLC) was used to determine the ascorbic acid content in the peel tissue. 0.5 g of tissue was homogenized in 2.0 mL of oxalic acid (w / v, g / 100 ml, 0.1%) and then centrifuged at 50000 g for 15 min at 4 °C. Subsequently, the supernatant was filtered through a 0.45 μm filter and the AsA content was determined using an Ultimate 3000 high performance liquid chromatography (HPLC) system. 20 mg·L -1 T-AsA was measured after addition of dithiothreitol (DTT), and the difference between T-AsA and AsA was taken as the DHA content.

[0100] Experimental results: PbrDHAR5 is located in the cytoplasm and nucleus (e.g. Figure 3 ). Transient overexpression of the PbrDHAR5 gene significantly increased cytDHAR activity in pear and tomato peel tissues, thereby increasing the AsA redox state; the opposite phenomenon was observed in the peel of the fruit in which the PbrDHAR5 gene was transiently silenced (e.g. Figure 3 ). Further research found that overexpression of the PbrDHAR5 gene significantly improved the resistance of tomato fruit to low temperature.

[0101] Example 3 Screening of upstream regulatory genes of PbrDHAR5 and its regulatory mechanism

[0102] 1. Screening of upstream regulatory genes of PbrDHAR5

[0103] Transcriptome analysis revealed a total of 38 differentially expressed TFs; of these, 6 were strongly positively correlated with PbrDHAR5 expression (fold change ≥ 1.5, FDR < 0.05; correlation coefficient ≥ 0.8). Since Pbr008639.1 had the highest correlation with PbrDHAR5 gene expression, it was named PbrWRKY83 and further studied. Using the PlantCARE database, the W-box elements in the promoter of the PbrDHAR5 gene (1502 bp sequence upstream of the transcription start site (ATG)) were analyzed and it was found that there were two W-box elements in the promoter of the PbrDHAR5 gene ( Figure 4 c). Further analysis of the PlantRegMap database revealed that PbrWRKY83 may interact with these two W-box elements to regulate the expression of the PbrDHAR5 gene.

[0104] 2. Dual luciferase reporter assay

[0105] The PbrWRKY83 gene coding sequence was cloned using the fruit cDNA of 'Dangshan Pear' as a template (primer sequences are shown in Table 1 in the attached table) and inserted into the pSAK277 vector (pSAK277-PbrWRKY83). The CDS sequence of the PbrWRKY83 gene is shown in SEQ ID No.3; the protein sequence encoded by the PbrWRKY83 gene is shown in SEQ ID No.4.

[0106] Using the fruit DNA of 'Dangshan Pear' as a template, the promoters of the PbrDHAR5 gene (PbrDHAR5pro, PbrDHAR5propro) containing different numbers of W-box elements (P1: (-453)-(-488); P2: (-1233)-(-1228)) were cloned. frag1 and PbrDHAR5pro frag2) (primer sequences are shown in Table 1 in the attached table), inserted into the pGreenII 0800-LUC vector, and constructed a reporter vector (pGreenII 0800-PbrDHAR5pro-LUC (full-length promoter), pGreenII 0800-PbrDHAR5pro frag1 -LUC, pGreenII 0800-PbrDHAR5pro frag2 -LUC). On this basis, mutant primers were designed (primer sequences are shown in Table 1 in the attached table) to mutate the W-box element in the promoter of the PbrDHAR5 gene (TTGACC / GGTCAA→TTTAGC / GCTAAA) to obtain the mutant promoter sequence PbrDHAR5pro mut , inserted into pGreenII 0800-LUC vector, and constructed reporter vector pGreenII 0800-PbrDHAR5pro mut -LUC.

[0107] The transcription factor recombinant plasmid (pSAK277-PbrWRKY83) and the reporter vector (pGreenII0800-PbrDHAR5pro-LUC or pGreenII 0800-PbrDHAR5pro frag1 -LUC or pGreenII 0800-PbrDHAR5pro frag2 -LUC or pGreenII 0800-PbrDHAR5pro mut -LUC) were transformed into Agrobacterium GV3101 and GV3101 (psoup) respectively. The positive strains were selected and expanded to OD 600 When the pH was 0.6-0.8, the bacteria were collected, and then the bacterial solution was resuspended with infection solution (10mMMgCl2, 10mM MES, 200μM acetosyringone, pH=5.7). After induction for 3-5h under dark conditions, tobacco leaves were infected. Before injection, the Agrobacterium solution containing pSAK277-PbrWRKY83 and the reporter vector were mixed at a ratio of 9:1 (v / v), and the tobacco leaves co-transfected with the Agrobacterium solution carrying the pSAK277 empty vector and the corresponding reporter vector were used as controls. 60-72h after infection, the leaves were collected and the activities of firefly luciferase and Renilla luciferase were detected using the Dual-Luciferase Reporter Assay System kit (Promega, USA) (see the instructions for specific steps).

[0108] 3. Yeast one-hybrid assay (Y1H)

[0109] Using the fruit cDNA of 'Dangshan Pear' as a template, the coding sequence of the PbrWRKY83 gene was cloned (primer sequences are shown in Table 1 in the attached table) and inserted into AD to form the prey vector PbrWRKY83-AD. Using the fruit DNA of 'Dangshan Pear' as a template, according to the distribution of W-box elements (P1: (-453)-(-488); P2: (-1233)-(-1228)), a 200-bp promoter fragment of the PbrDHAR5 gene containing wild-type W-box elements and its mutants was inserted into the pAbAi vector (primer sequences are shown in Table 1 in the attached table) to form the bait vector (PbrDHAR5pro S1 -pAbAi, PbrDHAR5pro S2 -pAbAi, PbrDHAR5pro S1mut -pAbAi and PbrDHAR5pro S2mut -pAbAi).

[0110] The bait vector and prey vector were co-transformed into yeast strains, and the Y1H experiment was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Weidi, Shanghai, China) (see the instructions for specific steps). PbrDHAR5pro was performed on SD / -Ura deficiency medium supplemented with aurobasidin A (AbA). S1 -pAbAi, PbrDHAR5pro S2 -pAbAi, PbrDHAR5pro S1mut -pAbAi and PbrDHAR5pro S2mut-pAbAi self-activation detection, and select the appropriate AbA concentration for subsequent experiments. The yeast strain co-transformed with the bait vector and the prey vector was cultured on a SD / -Leu deficiency medium supplemented with an appropriate concentration to detect the binding ability of PbrWRKY83 to the PbrDHAR5 promoter. The yeast strain co-transformed with pGAD-p53 and p53-AbAi (reference Jia L, Zhang X, Zhang Z, Luo W, Nambeesan SU, Li Q, Qiao X, Yang B, Wang L, Zhang S. PbrbZIP15 promotes sugar accumulation in pear via activating the transcription of the glucoseisomerase gene PbrXylA1. Plant J. 2024, 117 (5): 1392-1412.) was used as a positive control, and AD and PbrDHAR5pro S1 -pAbAi (or PbrDHAR5pro S2 -pAbAi, or PbrDHAR5pro S1mut -pAbAi, or PbrDHAR5pro S2mut -pAbAi) co-transformed yeast strain served as a negative control.

[0111] 4. ChIP-qPCR Experiment

[0112] Callus tissue of 'Clapp's Favourite (CF)' pear flesh was used as the experimental object. Wild-type callus tissue was grown on MS medium (30 g / L sucrose, 1 mg / L 2,4-D and 0.5 mg / L 6-BA) and subcultured every 2 weeks to prepare materials for subsequent transgenic. Callus tissue was infected with Agrobacterium carrying pCAMBIA1300-PbrWRKY83-GFP (the sequence of primers used to construct pCAMBIA1300-PbrWRKY83-GFP vector is shown in Table 1 in the attached table). The infected callus tissue was co-cultured on MS medium containing acetosyringone (AS) for 2 days, and then transferred to MS medium containing hygromycin (Hyg) (carbon source: 30 g / L sucrose) for 1.5-2 months. After DNA and RNA identification of regenerated callus tissue, positive lines were obtained and propagated. Subsequently, the positive callus transformed with the PbrWRKY83 gene and the control (callus transformed with no pCAMBIA1300-GFP) were co-transferred to MS medium (containing Hyg) with a mixture of sorbitol and sucrose (15 g / L each) or glucose (30 g / L) as the carbon source for subsequent determination.

[0113] 10 g of callus containing pCAMBIA1300-PbrWRKY83-GFP was weighed, crushed, and cross-linked in 1% (v / v) formaldehyde cross-linking buffer for 15-30 min. Glycine was added to terminate the cross-linking, and the cross-linked product was ground with liquid nitrogen and lysed to obtain chromatin, which was further ultrasonically broken to obtain soluble sheared chromatin (average DNA length 200-500 bp). A portion of the soluble sheared chromatin was used as input, and the remaining portion was washed and pre-cleaned for immunoprecipitation (IP) with GFP antibody. Pear callus carrying pCAMBIA1300-GFP was used as a control. Specific fluorescent quantitative primers were designed in the promoter region of PbrDHAR5 containing W-box elements (P1: (-453)-(-488); P2: (-1233)-(-1228)) (primers are shown in Table 1 in the attached table), and the enrichment multiple was detected by qPCR.

[0114] 5. EMSA Experiment

[0115] The full length of PbrWRKY83 CDS was amplified using the fruit cDNA of 'Dangshan Pear' as a template and inserted into the pCold-TF expression vector (primer sequences are shown in Table 1 in the attached table). The correctly sequenced pCold-PbrWRKY83 plasmid was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant protein His-PbrWRKY83 with a His tag, and the pCold-TF empty protein was expressed as a control. The crude protein was purified and used for subsequent experiments. According to the sequence information of the W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228)) on the promoter of the target gene PbrDHAR5, a DNA probe of about 30-40p was synthesized, including a biotin-labeled probe carrying an unmutated (or mutated) W-box element and an unlabeled competitive probe carrying a W-box element. The DNA probe was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (probe sequences are shown in Table 1 in the attached table). EMSA experiments were performed according to the manual provided by Chemiluminescent EMSA Kit (Biyuntian, China).

[0116] Experimental results: According to the results of transcriptome analysis, the expression levels of PbrWRKY83 and PbrDHAR5 genes were highly correlated, and they were taken as the objects of further research (such as Figure 4 PbrWRKY83 activates the expression of PbrDHAR5 gene by binding to the W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228)) in the upstream promoter of PbrDHAR5 gene (e.g. Figure 5). The P1 binding site sequence is (+ / -)TTGACT / AGTCAA; the P2 binding site sequence is (+ / -)TTGACT / AGTCAA.

[0117] Example 4 Functional verification of PbrWRKY83

[0118] 1. Subcellular localization of the protein encoded by the PbrWRKY83 gene

[0119] The coding sequence of PbrWRKY83 gene (excluding the stop codon) was amplified using the fruit cDNA of 'Dangshan Pear' as a template (primer sequences are shown in Table 1 in the Appendix), inserted into the pBI221-GFP vector, and transformed into tobacco leaves. AtH2B-mcherry was used as a nuclear marker, and the fluorescence signal was observed using a confocal microscope (Leica Microsystems, Germany).

[0120] 2. Transient overexpression of PbrWRKY83 gene in pear peel

[0121] Using the fruit cDNA of 'Dangshan Pear' as a template, the coding sequence of the PbrWRKY83 gene was amplified (primer sequences are shown in Table 1 in the attached table), inserted into the pCAMBIA1300 plasmid, and transformed into Agrobacterium GV3101. After PCR identification, positive strains were selected for propagation and cultured until OD 600 When the OD value was 0.6-0.8, the cells were collected; the cells were resuspended in infection solution (10 mM MgCl2, 10 mM MES and 200 mM acetosyringone), and after induction for 4 h under dark conditions, the OD value was adjusted. 600 The bacterial solution was injected into the fruit skin tissue of Dangshan Pear slowly and evenly with a syringe, and the fruit skin tissue was protected from light for 24 hours after injection. The fruit skin tissue at the injection site was collected 3 days after injection for subsequent determination. At the same time, fruits of similar size without pests and mechanical injuries were selected and injected with Agrobacterium carrying pCAMBIA1300 as a control.

[0122] 3. Transient silencing of the PbrWRKY83 gene in pear peel

[0123] Using the fruit cDNA of 'Dangshan Pear' as a template, the coding sequence of the PbrWRKY83 gene of about 250 bp was amplified (primer sequences are shown in Table 1 in the attached table), inserted into the pTRV2 vector, and transformed into Agrobacterium GV3101. After PCR identification, positive strains were selected for propagation and cultured until OD 660 The cells were collected when the OD was 1.0; the cells were resuspended in infection solution (10 mM MgCl2, 10 mM MES and 200 mM acetosyringone), and after 4 h of dark induction, the OD was adjusted. 660to 1.0. The bacterial solutions carrying the recombinant plasmids pTRV2-PbrDHAR5 and pTRV1 were mixed at a volume ratio of 1:1 and injected into the peel tissue of 'Dangshan Pear'. After injection, the mixture was kept away from light for 24 hours. The flesh of the injected part was collected 3 days after injection for subsequent determination. The peel tissue co-injected with bacterial solutions carrying pTRV2 empty vector and pTRV1 was used as a control.

[0124] 4. Obtaining transgenic tomato fruits and testing low temperature resistance

[0125] The constructed pCAMBIA1300-PbrWRKY83-GFP overexpression vector was transferred into Agrobacterium GV3101 by Agrobacterium-mediated genetic transformation, and tomato cotyledons were infected. Tissue culture seedlings were obtained by callus induction, resistance induction differentiation and rooting culture. The T2 generation overexpression seeds were tested for hygromycin resistance, and the positive transgenic homozygous strains were verified by PCR technology. The seeds of the T2 homozygous strain were planted, and the fruits were harvested 35 days after flowering, refrigerated at 4°C for 10 days and then placed at 20°C for 7 days. The wild-type tomato fruits 35 days after flowering were used as the control, and the chilling injury index was calculated according to the fruit maturity stage = Σ (number of fruits × fruit maturity stage) / total number of surveyed fruits (0 = red maturity stage, 1 = pink stage, 2 = color change stage, 3 = color breaking stage, 4 = green maturity stage).

[0126] 5. Analysis of PbrWRKY83 and PbrDHAR5 gene expression in tissues (qRT-PCR)

[0127] Total RNA was extracted from the tissue using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.), and the extracted total RNA was reverse transcribed to synthesize cDNA. The expression levels of PbrWRKY83 and PbrDHAR5 genes were detected using Light Cycler 480SYBR GreenIMaster (4887352001, Roche) reagent. The primer sequence of PbrDHAR5 gene is shown in Table 1 in the attached table, and the primer sequence of PbrWRKY83 gene is shown in Table 1 in the attached table. PbrTub was used as an internal reference gene.

[0128] 6. Enzyme Activity Assay

[0129] 0.2 g of sample was homogenized in the corresponding extraction buffer and then centrifuged at 50000 g for 15 min. After collecting the supernatant, cytosolic dehydroascorbate reductase (cytDHAR) (DHAR-1-w, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) was determined according to the formula of the relevant detection kit. The protein concentration in the supernatant was determined using a bicinchoninic acid protein detection kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0130] 7. Determination of ascorbic acid content in tissues

[0131] High performance liquid chromatography (HPLC) was used to determine the ascorbic acid content in peel tissue. 0.5 g of tissue was homogenized in 2.0 mL of oxalic acid (w / v, g / 100 ml, 0.1%) and then centrifuged at 50,000 g for 15 min at 4 °C. Subsequently, the supernatant was filtered through a 0.45 μm filter and the AsA content was detected using an Ultimate 3000 high performance liquid chromatography (HPLC) system. T-AsA was measured after adding 20 mg·L-1 dithiothreitol (DTT) to the supernatant. The difference between T-AsA and AsA was taken as the DHA content. Experimental results: PbrWRKY83 is located in the cell nucleus (such as Figure 6 ). Transient overexpression of PbrWRKY83 significantly increased PbrDHAR5 gene expression and cytDHAR activity in pear and tomato peel tissues, thereby increasing the AsA redox state; while the opposite phenomenon was observed when PbrDHAR5 gene was transiently silenced (e.g. Figure 6 ). Further research found that overexpression of the PbrWRKY83 gene significantly improved the resistance of tomato fruit to low temperature.

[0132] In summary, the present invention is based on a transcription factor PbrWRKY83, which can combine with the W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228)) in the promoter of the PbrDHAR5 gene to regulate its transcription level, affect the regulation of the redox state of AsA in pears, and thus enhance the fruit's resistance to low temperature stress (such as Figure 7 ), which has important theoretical and practical significance for breeding high-quality pear varieties resistant to tiger skin disease.

[0133] The above-mentioned specific implementation methods of the present invention do not constitute a limitation on the protection scope of the present invention. It should be pointed out that without departing from the technical principle of the present invention, any improvement, equivalent replacement, etc. made by those skilled in the art should be included in the protection scope of the claims of the present invention.

[0134] Schedule

[0135] Table 1 Primer or probe sequences related to subcellular localization, dual luciferase reporter assay, Y1H, ChIP-qPCR, EMSA, overexpression and gene silencing

[0136]

[0137]

[0138]

[0139] Sequence Listing

[0140] SEQ ID No.1

[0141] PbrDHAR5 CDS

[0142] Pyrus bretschneideri

[0143] ATGGTTCTCGAGGTCGCTGCCAAGGCTGCTGAGGGGCCCCCTGATCTCCTCGGCGAC

[0144] TGCCCTTTCTGCCAAAGGGTTACTCTAACCTTGGAGGAGAAGAAAGTACCCTACAAG

[0145] CTGCACCTCATCAATCTCAGCGACAAACCCAAATGGTTTACGGAAGTGAATCCAGA

[0146] GGGGAAGGTGCCTGTGGTGAAGTTTGATGACAAATGGGTGCCTGATTCCGATGTGA

[0147] TTGTTGGGATCATTGAGGAAAAATACCCTGAGCCTTCCTCAAAACCCCTCCTGAAT

[0148] TTGCTTCTGTGGGATCAAAGATATTGGGATCATTGTGACATTTCTGAAGAGCAAGG

[0149] ATCCCAGTGATGGATCAGAACAGGCTTTGCTCACGGAACTGAAGGCATTAGATGAG

[0150] CATCTTAAGGCACATGGTCCGTACATTGCTGGGGAGAAGGTCACTGCTGCGGATCTA

[0151] AGCTTGGCACCGAAACTGTACCATCTCAAGGTGGCTCTCGGCCATTTCAAGAAGTGG

[0152] ACGGTTCCAGCAGATTTGGCCCATTACCATAAGTACACCGAGCTGCTTTTCTCAAGG

[0153] GAATCTTTCGTGAAGACCGCTCCTGCTGACGAGAAATATATGATTGCAGGATGGGA

[0154] GCCGAAGGTGAATCCATGA

[0155] SEQ ID No.2

[0156] PbrDHAR5 Protein

[0157] Pyrus bretschneideri

[0158] MVLEVAAKAAEGAPDLLGDCPFCQRVTLTLEEKKVPYKLHLINLSDKPKWFTEVNPEGK

[0159] VPVVKFDDKWVPDSDVIVGIIEEKYPESLKTPPEFASVGSKILGSSFVTFLKSKDPSDGSE

[0160] QALLTELKALDEHLKAHGPYIAGEKVTAADLSLAPKLYHLKVALGHFKKWTVPADLAH

[0161] YHKYTELLFSRESFVKTAPADEKYMIAGWEPKVNP

[0162] SEQ ID No.3

[0163] PbrWRKY83

[0164] CDS

[0165] Pyrus bretschneideri

[0166] ATGAATGTGAAGAAGGATGTGGTGGAAGGGGAGGACAACATTAATAGCAACCATAT

[0167] GAATGGGGGAGGAGGAGGAGGTGGACTTACCACATCACCATTTGCAGGCATGTTTG

[0168] ATTTCTGTGAAGGAGAAAAGAGTTCATCTTTAGGGTTTATGGAACTTTTGGGGGTTG

[0169] GGGTTGGACAGGACTATTGTCCCACTTCCCTATTTGATTATCTGCCTCAGACACCATC

[0170] GACAGTCCTGCCTTCGTTAGCTTCCACAATGGGAGGAAAAGATCAGTTATCTACTGA

[0171] CTACTCCTCTTTGAATCAGCAGCCTACAACACCAAACTATTCGTCGATATCGTCGGA

[0172] ATCAAGCGAGGCAGTGAACGAAGAACACACTGATCATAAGGCTGCTACAGACCAAG

[0173] AGAAAAATAATGAAGAAGAAGAACATGACGAGCAACAAAAGACCAAGAAAGAGTT

[0174] GATAGCAAAGAAGGCGAGTCGGAAAAGGCAGAGAGAACCCAGATTTGCATTCATG

[0175] ACAAAGAGCGAAGTTGATCATCTGGAAGATGGCTACAGATGGAGAAAATACGGCCA

[0176] AAAAGCTGTCAAAAATAGCCCCTTTCCAAGGAGTTACTATCGGTGCACAAGTGCATC

[0177] ATGTAATGTGAAGAAGCGAGTGGAGCGATCTTTCGACGATCCGAGCATTGTTGTAA

[0178] CTACTTATGAAGGACAACACACTCATCGCAGCCCACTTCTGCCTCGCCCGACTCTCC

[0179] CTGCAGCTTCTGCTTTTCCAGCTAATAACTTTGCCATGCCATTAATCCCCACAAGAAC

[0180] CCTATTTACTCATGACTATCAACAAGTGCAACCATTTGCAGATAGCAACTCCTCTTC

[0181] GCCTTCATCGGCACCAATTAATTTTGGTGATTATTATGTGAATGGCTCTTCCTTGTGT

[0182] CTTACAAATGCTACTAGTACCGCTGGCTTTCTTCATCCCGAAAGGTGGTTTTGCTCCC

[0183] CGGCAACCGGTTCTGCTTTGTTAGCTGACCATGGCCTTCTTCCAAGACATAGTCCCCG

[0184] CTCATATGCTGAAGCAAGAGTAG

[0185] SEQ ID No.4

[0186] PbrWRKY83

[0187] Protein

[0188] Pyrus bretschneideri

[0189] MNVKKDVVKREDNINSNHMNGGGGGGGGLTTSPFAGMFDFCEGEKSSSLGFMELLGVG

[0190] VGQDYCPTSLFDYLPQTPSTVLPSLASTMGGKDQLSDTYSSLNQQPTTPNYSSISSESSEA

[0191] VNEEHTDHKAATDQEKNNEEEEHDEQQKTKKELIAKKASRKRQREPRFAFMTKSEVDH

[0192] LEDGYRWRKYGQKAVKNSPFPRSYYRCTSASCNVKKRVERSFDDPSIVVTTYEGQHTH

[0193] RSPLLPRPTLPAASAFPANFAMPLIPTRTLFTHDYQQVQPFADSNSSSPSSAPINFGDYY

[0194] VNGSSLCLTNATSTAGFLHPERWFCSPATGSALLADHGLLQDIVPAHMLKQSEQ ID No.5

[0195] PbrDHAR5 promoter

[0196] Pyrus bretschneideri

[0197] CACCGTCAGAGTCGCCGTTGCCATTGCCAACGGAATTGGATCTCATTTCCACATCTT

[0198] AGTTATTACTATGTATCATGCGGTTATAAATTATTTGATTTTTTTATTTAAAATTAA

[0199] ACACAAATAATATCTGATGAAAATTAATCGTACAATGTACAATTAACGAGGATGCA

[0200] GGGATTCCTAGAATCCCTACCAAGTAAATCCTAAGAGGATGCTCGGGTAACTATGTA

[0201] AATGGCGGTTGAAATAAGGGCCACGTGGTTATTAGTTATAGTCAACATTCGTACC

[0202] ATGTTCGTGGTGATTTGTAACATAGAAAATGATTAGAAAGTGTGATTAGAATGTAA

[0203] AGAGATGGAAATTTGAAGATCCACAATTTGTGGTTGAATGCTTGACACGTAAACG

[0204] ACCTAAATGGATAAATCAAATGTCCTTGCCAAAGATATAAACGGTAGTTGGAGAATT

[0205] AAATTCATTTTATTAATTATGGGAGAGAAAATTGGAATAATGGTCGTTTAATTTTA

[0206] ATCAAATTGAAGTTTTGATCCTTTAATTAAAAATTATTGATTATTGGTCTTTAACTT

[0207] ATTAAAATGTGCAGCTATGGTTATTTTTATCAACTTCGTTAGAACTTATATCAAAATA

[0208] AATCATGACGGAAGGACCTTTGTTATAATTGGTTAAAGTTGAGGGACCATTACTATA

[0209] ATTGGTTAAAGTTGAGGGATCATTGCTTTATTTGAGTTAAAGTTGAGGGACTATTGC

[0210] TAAAATTTTCTCATTTGATTTCCATATTTGCCACTTGATTGAGCCTCACGTACATGAC

[0211] ACGTGACTCATTAGACAAATTCTGACAGAGTTGATGAAAAGAATCATAAATGCACG

[0212] TTTTAAAGGACCAATAATCGTAGATTTTTAATTAAGAAATCATTACTCTAATTTAATT

[0213] AAATATGATCGACCATTGCTACAATTTCCTCTTAATGATAAATCAATTAACCACAGA

[0214] TCACATACCAATTCAAAGTCAGAGGCTTTTGAACACACACACACACACACACACAA

[0215] AAAAAAAAAAAAAAAAAAGATTAATTGACTGATTCTCTTTATAAACGCAGTTTTTG

[0216] CCGATGTACGTGTCTTTGAAAATTGATTATGTGCATTGTAAATGATGTAATTAACAC

[0217] TAACATGCTATTAAAATAATGAAAACTATTTCTACTTTATTAAACAAATAAAATTGT

[0218] GTCTTCTTTGAGGAAATTCAGAAACCATTTCTTTTCGAGAAGATGTTATCTTTATTTC

[0219] CCTTGTTAAATGATGTTGTATTTGTTTCATTACTGGTCTTGTTTTTCAGTTAAAATAA

[0220] GCCTTTTATATGCAAAGTGTATTAAGCTTGTGTCAGTACTGACATATGCCTTAATATA

[0221] TATATATATAGAGAGACACACACACACACACACACACCCCCTCAAAGCTCATCAAC

[0222] TCTCGCTCCATAACGAACCGTTCTGCCCCATTACCGTTCAGTTTCATCTTCCACACTC

[0223] TCTACCCCCTGGGCTCTCTA。

Claims

1. Application of PbrDHAR5 gene or biological materials related to PbrDHAR5 gene in regulating the redox state of ascorbic acid and thus enhancing the ability of fruit to resist low temperature stress. The nucleotide sequence of the PbrDHAR5 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The biological material related to the PbrDHAR5 gene is at least one of (a1) to (a7): (a1) PbrDHAR5 protein with an amino acid sequence as shown in SEQ ID NO.2; (a2) an expression cassette containing the PbrDHAR5 gene; (a3) a recombinant vector containing the PbrDHAR5 gene, or a recombinant vector containing the expression cassette described in (a2); (a4) a recombinant microorganism containing the PbrDHAR5 gene, or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3); (a5) a transgenic plant cell line containing the PbrDHAR5 gene, or a transgenic plant cell line containing the expression cassette described in (a2), or a transgenic plant cell line containing the recombinant vector described in (a3); (a6) transgenic plant tissue containing the PbrDHAR5 gene, or transgenic plant tissue containing the expression cassette described in (a2), or transgenic plant tissue containing the recombinant vector described in (a3); (a7) A transgenic plant organ containing the PbrDHAR5 gene, or a transgenic plant organ containing the expression cassette described in (a2), or a transgenic plant organ containing the recombinant vector described in (a3).

3. The use according to claim 2, characterized in that: Overexpression of the PbrDHAR5 gene regulates the redox state of ascorbic acid and thereby enhances the fruit's resistance to low temperature stress.

4. Application of WRKY family transcription factor PbrWRKY83 or biological materials related to WRKY family transcription factor PbrWRKY83 in regulating the redox state of ascorbic acid and thereby enhancing the ability of fruit to resist low temperature stress. The amino acid sequence of the transcription factor PbrWRKY83 is shown in SEQ ID NO.

4.

5. The use according to claim 4, characterized in that: The biological material related to the WRKY family transcription factor PbrWRKY83 is at least one of (b1) to (b7): (b1) gene PbrWRKY83 encoding the WRKY family transcription factor PbrWRKY83; (b2) an expression cassette containing the gene PbrWRKY83 described in (b1); (b3) a recombinant vector containing the gene PbrWRKY83 described in (b1), or a recombinant vector containing the expression cassette described in (b2); (b4) a recombinant microorganism containing the gene PbrWRKY83 described in (b1), or a recombinant microorganism containing the expression cassette described in (b2), or a recombinant microorganism containing the recombinant vector described in (b3); (b5) a transgenic plant cell line containing the gene PbrWRKY83 described in (b1), or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3); (b6) transgenic plant tissue containing the gene PbrWRKY83 described in (b1), or transgenic plant tissue containing the expression cassette described in (b2), or transgenic plant tissue containing the recombinant vector described in (b3); (b7) A transgenic plant organ containing the gene PbrWRKY83 described in (b1), or a transgenic plant organ containing the expression cassette described in (b2), or a transgenic plant organ containing the recombinant vector described in (b3).

6. The use according to claim 7, characterized in that: The nucleotide sequence of the gene PbrWRKY83 encoding the WRKY family transcription factor PbrWRKY83 in (b1) is shown in SEQ ID No.

3.

7. The use according to claim 4 or 5, characterized in that: The WRKY family transcription factor PbrWRKY83 interacts with the PbrDHAR5 gene described in claim 1 to regulate the redox state of ascorbic acid and thereby enhance the fruit's resistance to low temperature stress.

8. The use according to claim 7, characterized in that: The WRKY family transcription factor PbrWRKY83 regulates the redox state of ascorbic acid by combining with the WRKY binding site (W-box element (P1: (-453)-(-488); P2: (-1233)-(-1228))) in the PbrDHAR5 gene promoter, thereby enhancing the fruit's resistance to low temperature stress; the nucleotide sequence of the PbrDHAR5 gene promoter is shown in SEQ ID NO.

5.

9. A method for enhancing the ability of Rosaceae fruit to resist low temperature stress, characterized in that: Adopting any one of the following methods (c1)-(c2) or a combination of two methods to promote ascorbic acid accumulation and thus enhance the fruit's resistance to low temperature stress; (c1) overexpressing the gene PbrWRKY83 encoding the transcription factor PbrWRKY83 or / and increasing the protein level or activity of the transcription factor PbrWRKY83; (c2) overexpressing the PbrDHAR5 gene or / and increasing the protein level or activity of the PbrDHAR5 protein encoded by the PbrDHAR5 gene; The nucleotide sequence of the gene PbrWRKY83 encoding the transcription factor PbrWRKY83 is shown in SEQ ID No.3; the amino acid sequence of the transcription factor PbrWRKY83 is shown in SEQ ID No.4; and the nucleotide sequence of the PbrDHAR5 gene is shown in SEQ ID No.

1.

10. The use according to any one of claims 1 to 8, the method according to claim 9, characterized in that: The fruit is the fruit of a fruit tree of the Rosaceae family or the fruit of a vegetable of the Solanaceae family.

Images